1. Field of the Invention
The present invention relates to pharmaceutical compositions containing nitroxide compounds useful in ameliorating the deleterious effects of toxic oxygen-related species in living organisms, and methods of using the same.
2. Description of Related Art
The utilization of oxygen by mammals carries both a blessing and a potential curse. The blessing is that all mammals require oxygen for life. The potential curse is that during the metabolism of oxygen, a variety of toxic oxygen-related species such as hydroxyl radical, (.OH); hydrogen peroxide, (H2O2); and superoxide, (O.2xe2x80x94) are produced. Left unchecked, these free radical species could undoubtedly damage cells. However, cells have evolved elaborate detoxification and repair systems to rid themselves of these potentially toxic and undesirable metabolic by-products: superoxide dismutase (SOD) can convert superoxide to H2O2, and catalase (CAT) can convert H2O2 to H2O.
Yet another means to detoxify H2O2 (and organoperoxides) is via the enzyme glutathione peroxidase (GPX), which with glutathione (GSH), also converts H2O2 to H2O. Glutathione transferase (GST), in addition to its ability to conjugate and inactivate drugs and xenobiotics, also possesses peroxidase activity and can detoxify H2O2. These systems represent the major detoxification pathways for oxygen-derived free radicals species; however, there are doubtless other systems that may provide protection including protein sulfhydryls and other thiol-related enzymes that could be involved in repair mechanisms.
Despite the efficiency of these enzymatic systems, there is a small xe2x80x9cleakagexe2x80x9d of toxic species beyond the biochemical defense network. Of particular importance is the ultimate fate of H2O2 should it escape detoxification. H2O2, itself an oxidant capable of damaging biologically important molecules, can also undergo reduction via ferrous complexes to produce .OH. This reaction (often referred to as Fenton chemistry) produces the highly reactive .OH, which in the order of 10xe2x88x929 seconds, can: 1) abstract electrons from organic molecules; 2) break chemical bonds; 3) initiate lipid peroxidation; and 4) react with another .OH to produce H2O2. It is not known whether chronic exposure to low level oxygen-derived free radical species is deleterious; however, it is postulated that the process of aging may be a manifestation of the organisms""s inability to cope with sustained oxidative stress. Many modalities used in cancer treatment including x-rays and some chemotherapeutic drugs exert their cytotoxicity via production of oxygen-related free radicals, thereby imposing an added burden to normal detoxification systems. Additionally, free radicals and toxic oxygen-related species have been implicated in ischemia/reperfusion injury, and have long been thought to be important in neutrophil-mediated toxicity of foreign pathogens. Likewise, free radical damage has been implicated in carcinogenesis. The term xe2x80x9coxidative stressxe2x80x9d has thus emerged to encompass a broad variety of stresses, some of which have obvious implications for health care.
There has been considerable interest in devising additional approaches, apart from inherent intracellular detoxification systems, to protect cells, tissues, animals, and humans from the toxic effects of any agent or process that imposes oxidative stress. In the past few years, experimental studies have indicated that enzymes such as catalase and superoxide dismutase, and agents such as allopurinol and metal chelating compounds, afford protection against oxidative stress. None of these approaches is at present being applied to humans.
The application of the nitroxides of the instant invention is novel in this respect, and affords several unique advantages. Although the group of chemical compounds called stable nitroxide spin labels has had extensive biophysical use, they have never been used as antioxidants. They exhibit low reactivity with oxygen itself. Being low molecular weight, uncharged, and soluble in aqueous solution, they readily cross into the intracellular milieu. Enzymes such as catalase and superoxide dismutase do not. Therefore, the nitroxides should be superior to catalase and superoxide dismutase in that they can exert protection inside the cell. They are active within the biological pH range of about 5 to 8. Nitroxides are not proteins; therefore, the possibility of antigenic stimulation is remote. Previous low molecular weight superoxide dismutase mimics have all been metal dependent. The current agents do not contain metals, and problems with dissociation constants and deleterious metal induced reactions are therefore avoided. These compounds are apparently non-toxic at effective concentrations, and their lipophilicity can be controlled by the addition of various organic substituents, facilitating targeting of the molecules to specific organs or organelles where toxic oxygen-derived species are generated, to regions which are particularly susceptible to oxidative damage, or to the brain, if this is so desired. Previous radiation protectors have been sulfhydryl-group dependent. The current agents do not have a sulfhydryl group. Finally, previous use of these types of compounds as magnetic resonance contrast agents does not relate to their instant application as antioxidants.
Accordingly, it is an object of the present invention to overcome the problems associated with the use of impermeable enzymatic detoxifying agents such as superoxide dismutase and catalase to protect living tissues from the deleterious effects of toxic products generated during oxygen metabolism. This is accomplished by providing a pharmaceutical composition containing the nitroxide compounds by any means, and methods for using the same as metal independent, low molecular weight antioxidant for use as:
1) Ionizing radiation protectants to protect skin, and to protect against mucositis, the effects of whole body radiation, and radiation-induced hair loss. Administration in these situations may be accomplished either via topical application as an ointment, lotion, or cream, intravenously or orally by pill or lozenge.
2) Protectants against increased oxygen exposures so as to avoid, for example, pulmonary adult respiratory distress syndrome (ARDS).
3) Protectants against oxygen-induced lenticular degeneration and hyaline membrane disease in infants, and against oxidative stress-induced cataracts. The compounds may also be used to protect against oxidative stress in patients undergoing oxygen therapy or hyperbaric oxygen treatment. Administration under these circumstances may be accomplished via various routes including, for example, the use of eye drops, aerosol inhalation, or intravenous injection.
4) Reperfusion injury protectants effective in treating cardiovascular phenomena such as myocardial infarction and strokes, pancreatitis, or intestinal ulceration; to protect patients receiving organ transplants, and in organ preservation solutions.
5) Protectants for use in animal or plant cell culture media to prevent cytotoxicity due to excessive oxidation, for use in media designed for culturing aerobic microorganisms, for use in stabilizing labile chemical compounds which undergo spontaneous degradation by generating free radicals, for use in neutralizing free radicals which catalyze chain elongation during polymer formation, thereby terminating polymer elongation, and for use as a stabilizer for foods or food additives such as colors and flavors, especially in foods preserved via radiation treatment.
6) Biological antioxidants to protect humans and animals against agents such as the herbicide paraquat. In this circumstance, the pharmaceutical composition may be administered, for example, via inhalation as an aerosol to a subject exposed to paraquat. In addition, plants may be protected against such agents by, for example, spraying before or after exposure to such compounds.
7) Protectants against the cytotoxic effects of chemotherapeutic agents.
8) Protectants against mutagenic and carcinogenic agents. Administration in this situation or in 6, above, may be accomplished via oral ingestion, or parenterally.
9) Anti-inflammatory agents effective against arthritic conditions. For this purpose, the compositions may be administered parenterally, intra-articularly, or via oral ingestion.
10) Aging retardants. Administration for this purpose may be accomplished orally such as via a tablet supplement to the diet or parenterally.
11) Oral or intravenous agents inducing weight reduction.
These and other objects are accomplished by providing
a biologically compatible composition, comprising: an effective amount of a compound of the formula 
wherein R1 is xe2x80x94CH3; R2 is xe2x80x94C2H5, xe2x80x94C3H7, xe2x80x94C4H9, xe2x80x94C5H11, xe2x80x94C6H13, xe2x80x94CH2xe2x80x94CH(CH3)2, xe2x80x94CHCH3C2H5, or xe2x80x94(CH2)7xe2x80x94CH3, or wherein R1 and R2 together form spirocyclopentane, spirocyclohexane, spirocycloheptane, spirocyclooctane, 5-cholestane, or norbornane; R3 is xe2x80x94Oxe2x80x94 or xe2x80x94OH, or a physiologically acceptable salt thereof which has antioxidant activity; and a biologically acceptable carrier.
Compounds which may be useful in the present invention also include any compound having a 
or a salt thereof. These compounds can be represented broadly by the formula: 
wherein R3 is as defined above, and R4 and R5 combine together with the nitrogen to form a heterocyclic group. The atoms in the heterocyclic group (other than the N atom shown in the formula) may be all C atoms or may be C atoms as well as one or more N, O and/or S atoms. The heterocyclic group preferably has 5 or 6 total atoms. The heterocyclic group may be preferably a pyrrole, imidazole, oxazole, thiazole, pyrazole, 3-pyrroline, pyrrolidine, pyridine, pyrimidine, or purine, or derivatives thereof, for example.
Further compounds which may be useful in the present invention also include those wherein R4 and R5 themselves comprise a substituted or unsubstituted cyclic or heterocyclic groups.
Still further compounds which may be useful in the present invention also include oxazolidine compounds capable of forming an oxazolidine-1-oxyl.
Yet further compounds which may be useful in the present invention also include metal-independent nitroxides.
The present invention is also directed to methods for treating the deleterious effects of harmful oxygen-derived metabolic products, as listed above.
Physiologically acceptable salts include acid addition salts formed with organic and inorganic acids, for example, hydrochlorides, hydrobromides, sulphates such as creatine sulphate salts, phosphates, citrates, fumarates and maleates. The compounds of the invention have been shown to exhibit little or no in vitro cytotoxicity at concentrations of from 1-5 mM for 5 hours.
The compounds of the present invention can be used for the treatment of the toxic effects of oxidative stress in a variety of materials, cells and mammals including humans, domestic and farm animals, and laboratory animals such as hamsters, mice, rats, monkeys, etc. It is contemplated that the invention compounds will be formulated into pharmaceutical compositions comprising an effective antioxidant amount of the compounds of formula (I) and pharmaceutically acceptable carriers. An effective antioxidant amount of the pharmaceutical composition will be administered to the subject or organism, human, animal, or plant, in a manner which prevents the manifestations of oxidative stress. The amount of the compound (I) and the specific pharmaceutically acceptable carrier will vary depending upon the host and its condition, the mode of administration, and the type of oxidative stress condition being treated.
In a particular aspect, the pharmaceutical composition comprises a compound of formula (I) in effective unit dosage form. As used herein the term xe2x80x9ceffective unit dosagexe2x80x9d or xe2x80x9ceffective unit dosexe2x80x9d is denoted to mean a predetermined antioxidant amount sufficient to be effective against oxidative stress in vitro or in vivo. Pharmaceutically acceptable carriers are materials useful for the purpose of administering the medicament, which are preferably non-toxic, and may be solid, liquid or gaseous materials which are otherwise inert and medically acceptable, and are compatible with the active ingredients. The pharmaceutical compositions may contain other active ingredients such as antimicrobial agents and other agents such as preservatives.
These pharmaceutical compositions may take the form of a solution, emulsion, suspension, ointment, cream, aerosol, granule, powder, drops, spray, tablet, capsule, sachet, lozenge, ampoule, pessary, or suppository. They may be administered parenterally, intramuscularly or subcutaneously, intravenously, intraarticularly, transdermally, orally, buccally, as a suppository or pessary, topically, as an aerosol spray, or drops.
The compositions may contain the compound in an amount of from 0.1%-99% by weight of the total composition, preferably 1-90% by weight of the total composition, more preferably 10%-80% by weight of the total composition, and even more preferably, 25%-50% by weight of the total composition. For intravenous injection, the dose may be about 0.1 to about 300 mg/kg/day. If applied topically as a liquid, ointment, or cream, the compound may be present in an amount of about 0.01 to about 100 mg/ml of the composition, more preferably in an amount of about 5 to about 75 mg/ml of the composition, and even more preferably in an amount of about 25 to about 50 mg/ml of the composition. For inhalation, about 0.1 to about 200 mg/kg body weight of the compound should be administered per day. For oral administration, the compound should be administered in an amount of about 0.1 to about 300 mg/kg/day.
The invention also provides a method for treating the effects of oxidative stress due to the production of harmful oxygen-derived species which comprises administering an effective antioxidant amount of the above-mentioned compound to a organism, material, mammal or human susceptible to oxidative stress. Such stress includes that due to oxidizing agents, increased oxygen exposure, oxygen-induced degeneration or disease, reperfusion injury, ionizing radiation, carcinogenic, chemotherapeutic, or mutagenic agents, aging, or arthritis.
Reperfusion injury may include myocardial infarction, strokes, pancreatitis, and intestinal ulceration, while oxidative stress due to increased oxygen exposure includes pulmonary adult respiratory distress syndrome. Other oxidative stresses amenable to treatment with the compounds of the instant invention include oxygen-induced lenticular degeneration, cataracts or hyaline membrane disease in infants, or oxidative stress occurring during oxygen therapy or hyperbaric oxygen treatment.
Finally, in a further aspect of the invention, the compound of the instant invention can be used to prolong the storage life of human or animal cells, tissues, or organs by contacting these materials with a storage solution containing an effective amount of such compound, or to induce weight reduction in humans or animals.
The terms xe2x80x9cbiologically compatiblexe2x80x9d refer to a composition which does not cuase any adverse effects to an organism to which it is applied. The composition is preferably free of toxic substances or other substances which would render it unsuitable for the intended use.
The term xe2x80x9cparenteralxe2x80x9d includes an administration by injection such as intravenous, intramuscular, or subcutaneous.